The present invention relates to sweet proteins. Specifically, this invention relates to Brazzein protein that has been modified to provide a candy-like taste with high potency.
The most widely used natural sweetener, sugar (sucrose), has significant problems associated with its use (especially causing weight gain by users). Many other sweeteners either have undesirable side effects or are deficient in certain respects. For example, aspartame loses its sweetness when exposed to elevated temperatures for long periods. This renders aspartame unsuitable for use in most baking applications.
Moreover, most existing artificial sweeteners have temporal sweetness profiles which do not adequately match that of sugar. For example, their sweetness may die out sooner or leave an undesirable after taste, and/or may be perceived sooner than sugar. It may therefore be desirable to mix an existing artificial sweetener with one or more other sweeteners having different temporal profiles (so as to create a mixed sweetener that more closely matches the overall temporal sweetness profile of sugar).
Only relatively few sweet proteins (as distinguished from sweet carbohydrates) have been found in nature. One protein that appears to be particularly promising is Brazzein (SEQ ID NO: 1). See also U.S. Pat. Nos. 5,326,580, 5,346,998, 5,527,555 and 5,741,537, PCT publications WO 94/19467 and 95/31547, and J. Caldwell et al., Solution Structure Of The Thermostable Sweet-tasting Protein Brazzein, 5 Nature Structural Biology 427-431 (1998), F. Assadi-Porter et al., Efficient Production Of Recombinant Brazzein, A Small, Heat-Stable, Sweet-Tasting Protein Of Plant Origin, 376 Arch. Biochem Biophys. 252-258 (2000), and G. Hellekant, Characterization And Chemical Modification Of Brazzein, A High Potency Thermostable Sweet Protein From Pentadiplandra Brazzeana, 18 Acta Botanica Yunnanica 123-133 (1996); F. Assadi-Porter et al., Correlation Of The Sweetness Of The Protein Brazzein With Patterns Of Hydrogen Bonds Detected By NMR Spectroscopy, 278 J. Biol. Chem. 31331-9 (2003); F. Assadi-Porter et al., Brazzein, A Small, Sweet Protein: Effects Of Mutations On Its Structure, Dynamics And Functional Properties, 30 Suppl. 1 Chem. Senses i90-i91 (2005). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
This protein is a 53 amino acid residue protein, but also sometimes appears in nature linked at its N terminal to Glu. In any event, it is particularly desirable because it is stable when subjected to the level of heat typically present during baking of foods. However, there are some concerns regarding its temporal profile, the amount of the sweetener needed for threshold perception, and the perceived nature of the sweetness.
As described in WO 00/61759, and U.S. Pat. No. 6,274,707, attempts were made to improve certain sweetness characteristics of Brazzein through the substitution of Ala or Arg in replacement for an existing amino acid, and/or the addition of Ala or Arg, and/or the truncation of an existing terminal amino acid, of Brazzein. Some of these changes increased sweetness potency, while others decreased it.
Similarly, in H. Izawa et al. Pept. Sci.: Present Future, Proc. Int. Pept. Symp., 1st (1999)(Ed. Y. Shimonishi) there was a description of Ala substitutions for certain amino acids of Brazzein, with some results showing increased sweetness, while others showed decreased sweetness.
In U.S. Pat. No. 7,153,535 there was a discussion of the replacement of particular residues with Lys or Asn as positively affecting sweetness.
In Z. Jin et al., Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness, 28 Chem. Senses 491-498 (2003); Z. Jin et al., Critical Regions For The Sweetness Of Brazzein, 544 FEBS Letters 33-37 (2003); and F. Assadi-Porter et al., Sweetness Determinant Sites Of Brazzein, A Small, Heat-Stable, Sweet-Tasting Protein, 376 Archives of Biochemistry and Biophysics, 259-265 (2000) there was discussion regarding the N and C termini of Brazzein being important for sweetness (e.g. deletion of one C terminal residue eliminated sweetness).
While these developments are of significant interest, there is still a need for the development of protein sweeteners that provide a highly potent sweetness, particularly when providing a candy-like sweetness.